Proteolytic mapping of kinesin/ncd-microtubule interface: nucleotide-dependent conformational changes in the loops L8 and L12.
We used a battery of proteases to probe the footprint of microtubules on kinesin and ncd, and to search for nucleotide-induced conformational changes in these two oppositely-directed yet homologous molecular motors. Proteolytic cleavage sites were identified by N-terminal microsequencing and electrospray mass spectrometry, and then mapped onto the recently-determined atomic structures of ncd and kinesin. In both kinesin and ncd, microtubule binding shields a set of cleavage sites within or immediately flanking the loops L12, L8 and L11 and, in ncd, the loop L2. Even in the absence of microtubules, exchange of ADP for AMPPNP in the motor active site drives conformational shifts involving these loops. In ncd, a chymotryptic cleavage at Y622 in L12 is protected in the strong binding AMPPNP conformation, but cleaved in the weak binding ADP conformation. In kinesin, a thermolysin cleavage at L154 in L8 is protected in AMPPNP but cleaved in ADP. We speculate that ATP turnover in the active site governs microtubule binding by cyclically retracting or displaying the loops L8 and L12. Curiously, the retracted state of the loops corresponds to microtubule strong binding. Conceivably, nucleotide-dependent display of loops works as a reversible block on strong binding.
Alonso
M C
MC
Molecular Motors Group, Marie Curie Research Institute, Surrey, UK.
van Damme
J
J
Vandekerckhove
J
J
Cross
R A
RA
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000818:Animals; D018983:DNA Footprinting; D029721:Drosophila Proteins; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008958:Models, Molecular; D010449:Peptide Mapping; D011487:Protein Conformation; D051381:Rats; D013552:Swine
D029721:Drosophila Proteins; C066696:ncd protein, Drosophila; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D016547:Kinesins